Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

N,N'-diacetylchitobiase (chitobiase) from the marine organism Vibrio harveyi is a highly stable reporter enzyme for gene fusions. This enzyme hydrolyzes the disaccharide chitobiose to N-acetyl glucosamine. The advantages of the reporter gene encoding chitobiase (chb) are: (i) that chitobiase and N-acetyl-beta-D-glucosaminidase activities are missing in E. coli strains, (ii) chitobiase can be monitored using blue/white colony indicator plates and (iii) convenient substrates for this enzyme are commercially available. The use of chitobiase as a reporter enzyme is generally applicable to the study of gene expression in those bacteria that do not contain N-acetyl-beta-D-glucosaminidases. We constructed plasmid vectors containing a multiple cloning site for producing in-frame fusions to chitobiase, the attP of lambda phase for movement into the bacterial chromosome for single-copy analysis, the gene encoding chloramphenicol acetyltransferase (cat), the pACYC184 origin of replication and the rrnBt1t2 terminator region upstream of the chb gene to prevent read-through from other promoters. In-frame fusions between the dnaA gene and chb were moved to the chromosome by site-specific recombination with the chromosomal attB site. These single-copy fusions were assayed for chitobiase to examine the effects of a deletion in the dnaA regulatory region.
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PMID:Chitobiase, a new reporter enzyme. 986 57

Previously published studies suggest that an alteration in hexosamine flux induces a state of insulin resistance in muscle, liver, and other cell types. Glucosamine also alters the expression of several genes through an effect on transcription factors such as Sp1. Since the anti-atherogenic protein apolipoprotein AI (apoAI) is positively regulated by insulin, at least partly through its effect on Sp1, we investigated the effect of glucosamine on apoAI gene expression in the hepatocyte cell line, HepG2. By 24 hours of treatment with 0.1, 1, or 3 mmol/L glucosamine, the amount of apoAI protein secreted into the culture media increased 1.8-fold, 5.5-fold, and 2.3-fold, respectively. The decline in apoAI secretion at the highest glucosamine levels may be due to toxicity since the percentage of cells able to exclude trypan blue was lower in this group than in control cells (98.5% +/- 1.5% in control cells v 89.2% +/- 2.1% in cells treated with 3 mmol/L glucosamine, P <.01). ApoAI mRNA levels increased 2.4-fold in hepatocytes treated with 1 mmol/L glucosamine for 24 hours (1,158.1 +/- 78.8 v 482.2 +/- 24.3 arbitrary integrator units [AIU], P <.02), suggesting that the increase in apoAI protein secretion was due, at least partly, to an increase in apoAI mRNA levels. However, glucosamine had no effect on apoAI gene transcription rate as measured by nuclear runoff analysis (3,155 +/- 46.0 in control cells v 3,181 +/- 30.0 AIU in glucosamine-treated cells). Similarly, apoAI promoter activity measured in HepG2 cell transfected with an apoAI reporter plasmid containing the full-length apoAI promoter including an insulin-responsive Sp1 binding site did not change with glucosamine addition. In this assay, the chloramphenicol acetyltransferase (CAT) activity was 12.4% +/- 3.1%, 10.1% +/- 2.4%, 9.8% +/- 2.0%, 9.7% +/- 2.2%, and 11.9% +/- 2.9% in cells treated with 0, 0.03, 0.1, 0.3, and 1 mmol/L glucosamine, respectively. The apoAI mRNA turnover studies showed that 1 mmol/L glucosamine treatment of HepG2 cells was associated with increased apoAI mRNA half-life, from 7.6 to 16.6 hours. These findings suggest that increases in apoAI gene expression by glucosamine occur primarily through stabilizing apoAI mRNA.
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PMID:Effect of glucosamine on apolipoprotein AI mRNA stabilization and expression in HepG2 cells. 1516 26